Traumatic and osteoporotic fractures, critical/large defects and nonunion represent a significant burden in health care and affects quality of life for these patients. Tissue engineering approaches show promise as bone substitutes, and have been particularly successful in vitro in a bioreactor environment. The major challenge of tissue engineered regeneration is to maintain viability of cells in vivo and to rebuild vascular networks capable of delivering oxygen and nutrients while removing waste products after the implantation. Recently, a new cell homing approach has shown promising results in recruiting endogenous cells and then regeneration without cell transplantation. To accelerate cell homing and maintain cell viability in vivo, functional bone fluid flow induced by mechanical loading has been shown to be a critical regulator in initiating and mediating bone surface and osteonal adaptation. Dynamic fluid flow through porous constructs will exert increased fluid shear stress to promote in vivo cell differentiation and mineralization. Using oscillatory pressurized marrow fluid flow and muscle-bone interface stimuli, small magnitude fluid pressure (10-60 mmHg) with relative high frequency and short daily duration (10min) was found to initiate new bone formation and mitigate increased intracortical porosities caused by disuse osteopenia. It is essential to establish a functional dynamic fluid flow environment within the in vivo porous bone large defect and maintain an active fluid flow for bone regeneration. Thus, we will examine the general hypothesis that functional mechanotransduction regulated by dynamic bone fluid flow, with optimized intensity and rate, is essential and responsible for in vivo tissue regeneration, cellular differentiation, and osteogenic mineralization in critical defect healing. The ultimate gol is to generate an oscillatory fluid pressure gradient in the critical defect and the scaffold, servng as an in vivo bioreactor to promote functional fluid flow, vascular circulation, and osteogenesis. The outcomes will improve our understanding of how an optimized fluid flow environment enhances cellular viability and mineralization, and the importance of mechanotransduction in tissue repair and regeneration particularly under in vivo conditions. It is expected that this project will provide a novel approach to regulate bone formation via in vivo fluid flow stimuli, an improve our knowledge of critical signals for dynamic mechanotransduction in accelerating bone formation and mineralization in tissue regeneration, which will be ultimately used for clinical tissue repair.